DESCRIPTION: The periodontal pathogen, Actinobacillus actinomycetemcomitans, expresses several complex multi-gene toxin systems that promote the negative interaction of this bacterium with human cells. These systems include genes required for invasion and genetic loci for a leukotoxin and a cytolethal-distending toxin (CDT). This impressive and extensive repertoire of potential virulence factors has not been found in other oral microbial pathogens. The focus of this application is a continuation of our studies of the CDT of A. Actinomycetemcomitans Y4. This toxin is composed of three gene products which self-assemble to form a tripartite complex or holotoxin. The active subunit of the holotoxin is a nuclease, which is functionally related to mammalian DNase I. The DNase I-like protein targets the nucleus of most eukaryotic cells where it causes extensive DNA fragmentation and chromatin damage. These activities lead to growth arrest at the end of the G2 phase of the growth cycle. The major objectives of this application are to understand how the various subunits of the CDT function to produce a biologically active toxin, to define the interactions of the CDT with cell lines that are relevant to the integrity of the human oral cavity and to isolate and use CHO cell mutants to study cytotoxicity. The Specific Aims are: (i) to determine the interrelationships of the cdtA, cdtB and cdtC gene products, (ii) to determine the differential effects of the CDT on cultured oral epithelial cells and fibroblasts, and (iii) to use CHO cell mutants to examine and characterize specific roles of cdt gene products in the various stages of intoxication. Natively assembled recombinant holotoxin and holotoxin assembled in vitro, from purified recombinant Cdt proteins, will be used in cytotoxicity and binding experiments to examine the functions of the subunits. The differential effects of the CDT on a recently immortalized human oral epithelial cell line and human periodontal ligament fibroblasts will be examined to define the specificity and host range of the CDT. In a novel approach, CHO cell mutants resistant to the activities of the various CDT subunits will be isolated and used to segregate the subunit activities for study. The long-term goal of our study is to gain insight into the role of the Actinobacillus actinomycetemcomitans CDT in the perturbation of normal cell functions that are important for maintaining the healthy status of the human oral cavity.